Novel nucleotide coenzymes



United States Patent ice Patented idlifli The term nucleoside is usedherein to include deoxy- 3,032,203 nuoleosides. Thus, nucleoside withinthe meaning of NOVEL NUCLEOTIDE; (FOENZYMES the present inventionincludes: Leon Goldman and Joseph William Marslco, Nanuet, (4) Thymidine(or thymine deOXyfibOSidC):

N.Y., and George Washington Anderson, Upper Saddle River, N.J.,assignors to American Cyanamid Com- 5 pany, New York, N.Y., acorporation of Maine No Drawing. Filed Feb. 10, 1960, Ser. No. 7,744

4 Claims. (Cl. 260-211.5)

The present invention relates to a novel method of preparing nucleotidecoenzymes of biological and medical significance.

In order to clarify the exact nature of the chemical compounds of thepresent invention a few definitions will first be given beforeproceeding with the description of th invention.

A nucleoside is an N-glycoside of 'a heterocyclic base, generally apyrimidine or a purine. Examples of nucleosides are:

(1) Adenosine (or adenine riboside):

A nucleotide is a phosphate ester of a nucleoside and may be anucleoside monophosphate or a nucleoside polyphosphate. Examples ofnucleoside monophosphates are:

(l1) Adenosine-'5 phosphate (or muscle adenylic acid or adenosinemonophosphate, the latter hereinafter termed AMP) (3) Cytidine-Sphosphate:

ff H O-F-0H 3 (4) Thymidine-S phosphate:

Examples of nucleoside polyphosphates are: (l) Adenosine-S' diphosphate(hereinafter termed ADP):

(2) Adnosine-S' triphosphate (hereinafter termed ATP):

Originally, the term nucleotide referred only to phosphate esters ofnucleosides. Today, the term nucleotide is applied to phosphate estersof N-glycosides of heterocyclic bases generally. The newer definition,thus, includes not only the simple nucleotides of the originaldefinition, but also the nucleic acids (polynucleotides) and suchsubstances as adenosine-S' triphosphate and nicotinamide nucleotide.Derivatives of riboflavin phosphate, although not glycosidic in nature,are commonly included among the nucleotides because of their similarityto, and association with, true nucleotides.

A nucleotide coenzyme is a compound including in its structure at leastone simple nucleotide moiety. The term nucleotide coenzyme is applied toa large and growing group of substances which are vital components ofmany enzyme systems involved in metabolic processes. Nucleotidecoenzymes function in association with specific proteins or apoenzymes,the complete enzyme system being made up of the combination apoenzymeplus coenzyme. Historically, the first nucleotide coenzyme discoveredwas cozyrnase, or diphosphopyridine nucleotide,

discovered by Harden and Young in 1904. Examples of nucleotide coenzymesare:

(1) Cozymase (or diphosphopyridine nucleotide):

(2) Flavin adenine dinucleotide:

Oil

(3) Uridine diphosphate glucose:

The preparation of adenosine-S' phosphoroimidazole from adenosine-5'phosphate, dicyclohexylcarbodiimide and imidazole was reported by R. W.Chambers and I. G. Moffatt [1. Am. Chem. Soc. 80, 3752 (1958)].Thereafter, the more facile preparation of nucleotide imidazoles by theinteraction of a salt of a nucleotide and a l,1-carbonyldiimidazole wasdiscovered by L. Goldman et a1. as is more fully set forth in U.S.Patent No. 2,951,838.

The reactivity of phosphorylated imidazoles is much greater than that ofthe ordinary phosphoramidates which also contain a phosphorus-nitrogenlinkage. It has been postulated that this is a result of electronicdisplacements associated in particular with the electron attraction ofthe unsubstituted nitrogen atom. Surprisingly, however, although thephosphorus-nitrogen bond in the nucleotide imidazoles is thus generallyactivated, it is nevertheless resistant to reaction with water. Hence,it is possible to prepare nucleotide coenzymes and related compoundssuch as the linear and cyclic oligonucleotides from the nucleotideimidazoles in the presence of water as a solvent without the startingmaterial reacting with that solvent. This NHZ.

O RENE RC 02H II rrlsol ll RCHCOzH Base-sugar-O ROH Base-sugar-O ROP0311 wherein the Base may be either a purine or a pyrimidine moiety, theSugar may be either a pentose or a hexose moiety such as D-ribose,D-glucose, or 2-deoxy-D-rib-ose, and wherein imidazole is a by-productin each case. These nucleotide coenzymes may be prepared by the novelmethod of the present invention in aqueous or nonaqueous media, attemperatures of from C. up to 100 C., and over a period of time of froma few minutes up to 12 hours.

The nucleotide coenzymes produced by the novel method of the presentinvention are useful as vital components of many enzyme systems. Theyare useful as organic catalytic agents in that they are capable ofaltering the velocity of many chemical reactions.

The following examples illustrate the novel method of preparingnucleotide coenzymes of the present invention.

EXAMPLE 1 Preparation of l,3-Dicycl0hexylguanidinium Azlenosine-b"Phosphoramia'ate A solution of adenosine-S' phosphoroimidazole inanhydrous dimethylformamide (prepared from 1.00 g. of adenosine-S'phosphate hydrate, 0.586 g. of imidazole, and 1.38 g. of1,l'-carbonyldiimidazole) was diluted with 2 N ammonium hydroxide andtert-butyl alcohol and heated in a stainless steel bomb at 92 C. for 11hours.

6 To the bomb contents, 0.612 g. of 1,3-dicyclohexylguanidine was addedand the resultant solution evaporated under reduced pressure to a gummyresidue. Addition of acetone followed by filtration gave 1.55 g. ofcolorless crystals of 1,3-dicyclohexylguanidinium adenosine-Sphosphoramidate (solvated with Water and dimethylformamide), meltingpoint 211-214 C. dec. The product was homogeneous has shown by paperchromatography in isopropyl alcohol-ammonia-water (7-1-2) and by paperelectrophoresis in pH 7.5 phosphate buffer. Recrystallization fromaqueous acetone gave l,3-dicyclohexylguanidinium NHZ adenosine-S'phosphoramidate, melting point 236238 C. dec.

EXAMPLE2 Preparation of 1,3-Dicycl0hexylguanidinium Adenosine-SPhosphora midate A solution of adenosine-S phosphoroimidazole in 7.5 ml.of anhydrous dimethylformamide (prepared from 1.00 g. of adenosine-Sphosphate hydrate, 0.586 g. of imidazole, and 0.92 g. of 1,lcarbonyldiimidazole) was dissolved in 20 ml. of 0.46 N ammoniumhydroxide and heated in a stainless steel bomb at 65 C. for 10 hours. Tothe bomb contents was added 0.612 gram of 1,3,- dicyclohexylguanidineand the solution was evaporated to dryness under reduced pressure.Recrystallization of the residue from aqueous acetone gave 1.41 g. ofcolorless crystals of 1,3-dicyclohexylguanidinium adenosine-Sphosphoramidate (solvated with water and dimethylform amide), meltingpoint 236-238 C. dec.

EXAMPLE 3 Preparation of Acridinium Aden0sine-5 Pyrophosphate To astirred solution of adenosine-S' phosphoroimidazole in anhydrousdimethylforrnamide (prepared from 0.200 g. of adenosine-S' phosphatehydrate,-0.ll7 g. of imidazole, and 0.0886 g. of1,1'-carbonyldiimidazole) at 10 C. to 20 C. was added dropwise asolution of 0.24 ml. of 85% phosphoric acid in dimethylformamide. Themixture was allowed to warm to room temperature during one-half hour.The gummy solid, which separated on chilling, was dissolved in dilutesulfuric acid and treated with ethanolic acridine to yield 0.222 gram ofacridinium adenosine-S' pyrophosphate as yellow crystals, melting point2l2-2l5 C. dec. Recrystallization from water gave yellow needles,melting point 216-217 C. dec. Paper chromatography in 5% disodiumphosphateisoamyl alcohol and in isopropyl alcohol-|1% ammonium sulfate(3-2), and paper electrophoresis in 0.02 M potassium dihydrogenphosphate, showed as the only impurity a trace of adenosine-S phosphate.

EXAMPLE 4 Preparation of P P -DiQdBIZOSiIIE-S' Pyrophosphate To astirred solution of 1.00 gram of adenosine-5 phosphate monohydrate and0.586 gram of imidazole in 7.5 ml. of anhydrous dimethylformamide at toC. was added 0.922 gram of 1,1-carbonyldiimidazole. After five minutesthe temperature of the reaction mixture was loweredo to -20 C., andafter 10 minutes 1.00 gram of adenosine-S' phosphate was added, withstirring. After minutes the reaction mixture was warmed to roomtemperature, and after 26 hours 3 ml. of pyridine was added. After 47hours the resulting solution was worked up by chromatography on Dowex-l(formate) [S. M. H. Christie, D. T. Elmore, G. W. Kenner, A. R. Rodd,and F. J. Weymouth, 1. Chem. Soc., 2947 (1953)] to yield, by evaporationof the 0.5 N formic acid eluate, 1.06 gram of P P -diadenosine-S'pyrophosphate sesquihydrate as colorless crystals, which werehomogeneous by paper chromatography in 5% disodium phosphateisoamylalcohol and in n-butyl alcohol-acetic acid-water (5-2-3), and by paperelectrophoresis in 0.02 M potassium dihydrogen phosphate.

8 EXAMPLE 5 Preparation of P -Aden0sine-5' P -UridiMe-S Pyrophosphate Asolution of adenosine-S' phosphoroimidazole in anhydrousdimethylformamide (prepared from 0.0903 g. of adenosine-5 phosphatehydrate, 0.0528 g. of imidazole, and 0.132 g. of 1,lcarbonyldiimidazole) was added to 0.1045 g. of imidazolium uridine-Sphosphate. The reaction was diluted with 1.0 ml. of anhydrous pyridineand stirred for 74 hours at room temperature. When an aliquot wasremoved and subjected to paper electrophoresis in an acetate buffer ofpH 4.8, the major component, P -adenosine-5 P -uridine-S pyrophosphate,traveled 15.7 cm. towards the anode, whereas the minor components,adenosine-5' phosphate, uridine-S' phosphate, and P P -(diuridine-S')pyrophosphate traveled 9.3, 13.0 and 18.7 cm., respectively.

We claim:

1. The method of preparing nucleotide coenzymes which comprises reactinga nucleotide imidazole with a nucleophilic agent selected from the groupconsisting of ammonia, primary amines, secondary amines, carboxylicacids, sulfuric acid, carbonic acid, N-blocked a-amino acids, alcohols,phosphate esters and phosphoric acid.

2. The method of preparing adenosine-S phosphoramidate which comprisesreacting adenosine-S phosphoroimidazole with aqueous ammonia at atemperature between C. and C.

3. The method of preparing adenosine-S pyrophosphate which comprisesreacting adenosine-fi' phosphoroimidazole with phosphoric acid at atemperature between -20 C. and 30 C.

4. The method of preparing P W-diadenosine-S' pyrophosphate whichcomprises reacting adenosine-S phosphoroimidazole with adenosine-S'phosphate at :1 mm perature between 20 C. and 30 C.

No references cited.

1. THE METHOD OF PREPARING NUCLEOTIDE COENZYMES WHICH COMPRISES REACTINGA NUCLEOTIDE IMIDAZOLE WITH A NUCLEOPHILIC AGENT SELECTED FROM THE GROUPCONSISTING OF AMMONIA, PRIMARY AMINES, SECONDARY AMINES, CARBOXYLICACIDS, SLUFURIC ACID, CARBONIC ACID, N-BLOCKED A-AMINO ACIDS, ALCOHOLS,PHOSPHATE ESTERS AND PHOSPHORIC ACID.